Chagas disease is caused by the protozoan parasite
Trypanosoma cruzi, widespread in the American Continent. A
recent report of the World Health Organization estimated that 16-18
million people are currently infected and that about 90 million
individuals living in endemic areas are at risk of contracting
T. cruzi infection. The high prevalence and continental
extension of Chagas disease in Latin America, as well as the
constant migration of T. cruzi infected individuals to
non-endemic areas, require efficient and practical diagnostic
procedures (Schmunis 1991,Wendel & Gonzaga 1993).

Chagas disease is characterized by the chronological appearance
of specific classes of antibodies during the development of the
infection. Antibodies from IgM class first appears as a typical
sign of the acute phase of the disease, and there are some reports
of an increase of total and specific IgA antibody class at this
early phase (Lorca et al. 1995, Umezawa et al. 1996a). Antibodies
of the IgG class, already present in the acute phase, accompanies
the infection until the chronic phase.

Detection of antibodies against T. cruzi antigens by
serological methods is still the main support for diagnosis of
Chagas disease. The commercial available diagnostic tests are based
on the whole or semi-purified antigenic fractions from T.
cruzi epimastigote (the non-infective forms of the parasite).
Considerable variation in the reproducibility and reliability of
these tests have been reported by different laboratories, mainly to
cross-reactivity with other pathogens and standardization of the
reagents (Camargo et al. 1986). Diagnostic tests employing
epimastigote antigenic extract have a limited specificity,
associated to the fact of they do not possess highly reactive
epitopes for IgG/IgM antibodies present in patients with acute or
congenital Chagas disease (Umezawa et al. 1996a-c). The
epimastigote antigenic fraction is constituted by complex
molecules, that favors the appearance of false positive reactions
and cross reaction with sera from patients with another infections,
mainly visceral leishmaniasis (Chiller et al. 1990). This antigenic
heterogeneity does not allow the differential diagnosis between the
acute and chronic phases, and also among the clinical
manifestations of Chagas disease.

EVALUATION OF RECOMBINANT ANTIGENS FOR CHAGAS DISEASE
SERODIAGNOSIS

As discussed above the commercial immuno-diagnostic tests are
based on the whole or semi-purified extracts of T. cruzi.
The lack of specific and well characterized antigens prepared under
quality-control conditions have introduced a source of variability
in the final reagent and controversial results have been obtained
with these reagents (Camargo et al. 1986). T. cruzi
recombinant antigens may provide a convenient tool to improve
current methods of serological diagnosis of Chagas disease (Moncayo
& Luquetti 1990, Levin et al. 1991, Krieger et al. 1992, Franco
da Silveira 1992, Paranhos et al. 1994, Pastini et al. 1994).
Cloned peptides carrying well defined epitopes can be produced on a
large scale and with acceptable degree of purity. For this reason,
we have decided to evaluate the diagnostic efficiencies of
different T. cruzi recombinant antigens in an ELISA format
(Umezawa et al. 1999). Six T. cruzi recombinant antigens
were expressed in fusion with glutathione S-transferase (GST) in
Escherichia coli and purified by affinity chromatography.
The selected antigens (H49, JL7, B13 and JL8, see
Table) are composed by tandem amino acid repeats and showed
high sensitivity, specificity, and positive and negative predictive
values in previous studies (Moncayo & Luquetti 1990, Levin et
al. 1991). Two non repetitive antigens A13 (Paranhos et al. 1990)
and 1F8 (Gonzalez et al. 1985) were also included in this
evaluation. The diagnostic efficiency of the ELISA using the six
recombinant antigens was carried out with 541 serum samples (304
samples from chagasic patients, and 237 samples from nonchagasic
patients) from nine South and Central America countries (Argentina,
Brazil, Bolivia, Chile, Colombia, El Salvador, Guatemala, Honduras
and Venezuela). The development and evaluation of the recombinant
ELISA was carried out by a central laboratory (E. Umezawa,
Instituto de Medicina Tropical da USP, SP, Brasil). The results
were compared to those obtained with the whole extract of
epimastigote forms.

Four recombinant antigens (1F8, H49, JL7 and B13) showed high
sensitivity varying from 93.4% to 99%. A large number of
individuals living in endemic areas produce specific antibodies
against repetitive amino-acid antigens (H49, JL7 and B13).
Interestingly, the sensitivity (99%) and specificity (99.6%) of 1F8
antigen is comparable to that other repetitive antigens indicating
that chronic chagasic patients also display antibodies against non
repetitive antigens. Serum samples from chagasic patients reacted
with, at least, one recombinant antigen, suggesting that a mixture
of recombinant antigens may detect anti-T. cruzi antibodies
in all of serum samples used in this study. The positivity of a
hypothetical antigenic mixture composed by the recombinant peptides
H49 or JL7, B13 and 1F8 was calculated as being 100%. Available
recombinant antigens react with 87-99% of the chronic chagasic
sera, suggesting that the combination of two or more antigens to
build up a multi-antigen immunoassay may result in a truly reliable
T. cruzi serodiagnostic test.

In this study the epimastigote antigen showed 100% of
sensitivity. However, the specificity of the epimastigote antigenic
extract was lower (84%) than recombinant proteins which displayed
specificity values between 96.2% (JL8) to 99.6% (A13, B13 and 1F8).
The lower specificity of the epimastigote antigen is mainly due to
cross-reacting epitopes between T. cruzi and
Leishmania sp. Our results indicate that one of the major
advantage of recombinant ELISA for the serodiagnosis of Chagas
disease is the lack of cross-reaction with other parasitic diseases
such as leishmaniasis. As it has been suggested by several authors
(Levin et al. 1991, Krieger et al. 1992, Pastini et al. 1994,
Umezawa et al. 1999), the use of a cocktail of recombinant antigens
should provide a reliable T. cruzi serodiagnostic test.
Different complementary antigens could be combined in a relatively
simple immunoassay, and lead to the development of a multi-antigen
diagnostic kit standardization for routine diagnosis of Chagas
disease.

As it was discussed above, the diagnosis of acute and congenital
Chagas disease, and the follow up of treated chagasic patients are
of paramount importance. So far, the unique test described to
control the parasitological cure of treated chagasic patients is
the lysis of bloodstream trypomastigotes mediated by complement
(Krettli et al. 1982). However, several groups point many
methodological difficulties for the inclusion of that test in the
routine of clinical laboratories (Pereira et al. 1989). For this
reason, the search of specific antigens and/or alternative tests
remains important for the follow up of the chagasic patients after
drug treatment (Almeida et al. 1991, Norris et al. 1994, Krautz et
al. 1994, Levy et al. 1996).

We have developed an immunodiagnostic test for Chagas disease
using the TESA liberated, as exoantigens, in the supernatant of
mammalian cell cultures infected with T. cruzi (Umezawa et
al. 1996c). Exoantigens were collected from supernatants of LLC-MK2
cell cultures, separated by SDS-polyacrylamide gel electrophoresis
and transferred onto nitrocellulose filters. The test was developed
by immunoblotting and termed TESA-blot. The main components of TESA
antigens are a 150-160-kDa protein and several components (130-200
kDa) of SAPA antigen (Shed Acute Phase Antigen), the last
previously described by Frasch and co-workers (Affranchino et al.
1989). The presence of SAPA/trans-sialidase in TESA-blot was
confirmed by reactivity with a specific antiserum against SAPA as
well as with IgG/IgM antibodies from acute and congenital human
sera (Umezawa et al. 1996c).

The evaluation of TESA-blot was carried out with serum samples
from 512 patients, 111 non-chagasic including cases of
leishmaniasis and other pathologies, and 401 chagasic patients
includind acute, congenital and chronic cases. TESA-blot showed
100% of sensitivity and 100% of specificity. Furthermore, this
assay allows the discrimination between acute and chronic phases of
Chagas disease. We have found that IgGs from chronic chagasic
patients reacted with the 150-160 kDa exoantigen whereas IgGs/IgMs
from acute patients reacted with the components of
SAPA/trans-sialidase. This test, until now, have been
demonstrated to be useful as a confirmatory test, since it has been
used to confirm several inconclusive results obtained by
conventional serology. Preliminary results from our laboratory
(Matsumoto & Umezawa) showed that a 160 kDa antigen, obtained
from DNA recombinant technology, may correspond to the CRP
(Complement Regulatory Protein), previously described by Norris et
al. (1994). Martins at al. (1985) had previously demonstrated that
reactivity with a 160-kDa trypomastigote surface protein could
discriminate between cured treated patients from the non cured
ones. For these reasons, we started to use these antigens in the
follow up of chagasic patients submitted to chemotherapy.

This work was supported by grants from Fapesp (96/06736-1),
CYTED (Ibero American Project of Biotechnology), International
Atomic Energy Agency (IAEA), FMUSP-LIM49 and CNPq.